The field of specific binding assays has greatly expanded as its importance in the diagnostic field has become recognized. The ability to detect a specific compound and measure the compound quantitatively has permitted the monitoring of the administration of a wide variety of drugs, the determination of an imbalance in a wide variety of hormones, the quantitation of physiologically active proteins and the diagnosis of the presence of a pathogen. Different techniques have been distinguished in requiring or not requiring separation steps, the nature of the signal developed by the label, the development of the signal in a solution or on a surface and the manner of measurement for quantitative determination.
In developing an assay, there are a number of considerations in devising the reagents and protocol. One consideration is the degree of sophistication of the individual performing the assay. There are a lot of situations where it is desirable that a relatively untrained or unexperienced individual should be able to perform an assay and obtain reasonably quantitative results. It is particularly desirable that the untrained person be able to perform a quantitative assay with a rapid simple test without the need for sophisticated instruments.
In the last decade an enormous amount of so-called dipsticks and filter assays have been developed varying from all kinds of paper strips in different shapes, promising a better result than a previous strip, to plastic strips, coated with, for instance, an immunochemical component.
For instance, European patent application EP 0,149,168 describes an immunoassay which can be carried out by making use of a capillary glass tube. At least 2 regions are packed with separate carrier material. An immunoreactive component provided with a labelling substance which, via an immunochemical reaction, is capable of forming an immunocomplex with a substance, whose presence or concentration in the test liquid is desired to determine, is bound to the first carrier material. Subsequently, said complex is transported by capillary action and ends up in the second carrier material where it is immobilized after binding to a second immunoreactive component which is bound to the second carrier material. Thereafter the quantity of immunocomplex, thus immobilized, can be measured via known detection methods depending on the labelling substance used. The labelling substances used in the European application in question are radioisotopes, enzymes or fluorescent substances.
A disadvantage of said immunoassays is that after the immunochemical reaction, several operations always have to be performed to separate bound solid phase from non-reacted reactants, which action is known to those skilled in the art as "bound/free separation". Additional operation steps are needed to add reagents after bound/free separation, which is for instance the case when a substance has to be added to detect a solid phase bound labelled reactant. An additional step is needed for certain when applying highly sensitive assays requiring enhancement of the assay signal.
Surprisingly, a device has now been found with which the operations to be performed remain confined to bringing a device into contact with a test liquid and a wash fluid and determining the result--after some time--without impairing the accuracy and reliability, making it possible also to perform complex assays, e.g. ELISA, with only two operation steps, irrespective the number of bound/free separations and reagent additions and the sequence thereof.